The oil-crisis In the early 1970's started an intense search for an alternative to petrol as automobile fuel. Ethanol emerged as a good candidate since it to a large extent can replace petrol without major changes of combustion engines. It can be produced from renewable, lignocellulosic biomass such as agricultural and forest residues. Unlike petrol, ethanol produced from a renewable resource, does not give a net contribution of carbon dioxide to the atmosphere and would therefore not contribute to global warming. Sweden aims to substitute 15% petroleum-based fuels with fuels derived from renewable resources by 2010 (Kommunikationskommitén, 1996).
Furthermore, ethanol can replace some of the fuel (gasoline) in today's engine without any adjustments at all, or at least very small adjustments.
Lignocellulosic material mainly contains cellulose, hemicellulose and lignin. On average, wood dry-weight consists of 40% cellulose, 18% hemicellulose and 22% lignin (Taherzadeh, et al., 1997). Cellulose is composed of glucose residues while hemicellulose is a heteropolymer consisting of hexoses (mannose and galactose) and pentoses (xylose and arabinose). Lignin is a heterogeneous aromatic polymer made up of phenylpropanoid precursors.
To liberate the fermentable hexoses and pentoses, the lignocellulosic material is hydrolysed (Saddler, et al., 1993; Stenberg, et al., 1998; Tengborg, et al., 1998). During hydrolysis, fermentation-inhibiting substances like phenolics, furan derivatives (furfural and hydroxymethyl furfural) and acids (acetic, formic and levulinic acid) are formed from components in the lignocellulosic material (Larsson, et al., 1999; Palmqvist and Hahn-Hägerdal, 2000a; Palmqvist and Hahn-Hägerdal, 2000b).
Ethanol is a low value product where the raw material accounts for a large fraction of the total cost. Hence efficient utilisation of raw material is of crucial importance for an economically feasible process (Hinman, et al., 1989; von Sivers and Zacchi, 1996). An ideal microorganism to use for the fermentation of lignocellulosic material should i) have a broad substrate range and ferment all sugars to ethanol, preferably with high yield and productivity and ii) survive in a hydrolysate with fermentation inhibitors.
Microorganisms considered for fermentation of lignocellulosic material include Escherichia coli (Moniruzzaman, et al., 1996), Klebsiella oxytoca (Moniruzzaman, et al., 1996), Zymomonas mobilis (Bothast, et al., 1999), Pichia stipitis (Ferrari, et al., 1992) and Saccharomyces cerevisiae (Björling and Lindman, 1989; Olsson and Hahn-Hägerdal, 1993; Moniruzzaman, et al., 1996; Taherzadeh, et al., 1999; Hahn-Hägerdal, et al., 2001). Man has used the yeast S. cerevisiae for baking and production of alcoholic beverages for thousands of years. S. cerevisiae grows anaerobically on glucose (Andreasen and Stier, 1953; Andreasen and Stier, 1954; Visser, et al., 1990) and produces ethanol from glucose with high yield and high productivity (Kolot, 1980). Furthermore, S. cerevisiae is adapted to high ethanol concentrations (Jones, 1989) and has better tolerance towards fermentation inhibitors compared to bacteria (Olsson and Hahn-Hägerdal, 1993) and other yeast (Björling and Lindman, 1989; Olsson and Hahn-Hägerdal, 1993). However, unlike bacteria and several yeast strains (Skoog and Hahn-Hägerdal, 1988), wild-type S. cerevisiae can not utilise pentoses like xylose and arabinose. Still, after the introduction of genes encoding enzymes catalysing steps in initial pentose utilisation, growth and ethanol yield of recombinant S. cerevisiae have been poor. This thesis describes my efforts to analyse xylose utilisation by recombinant S. cerevisiae and Improve its xylose utilisation ability.
Metabolic engineering to improve xylose utilisation in S. cerevisiae 
With the introduction of recombinant DNA technology it has become possible to clone genes from one organism and transfer them to another organism, delete genes in the genome and also vary the expression levels of genes. It is thus possible to perform directed modifications of metabolic pathways. This new discipline is called metabolic engineering and has been defined as “Improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology” (Bailey, 1991) and “Purposeful modification of intermediary metabolism using recombinant DNA techniques” (Cameron and Tong, 1993). Like all classic fields of engineering, metabolic engineering is characterised by an analysis step and a synthesis step (Bailey, 1991; Stephanopoulos, 1994; Nielsen, 1998). In the analysis step, the microorganism is physiologically characterised and evaluated using, for instance, MFA (metabolic flux analysis), enzymatic activity measurements or expression analysis (Nielsen, 2001). The analysis provides information on where genetic modifications may improve the performance of the microorganism. The synthesis step involves the construction of a strain, with genetic modifications based on the analysis, using recombinant DNA technology. The new recombinant strain is then analysed using the same methodology as for its parental strain. If the analysis reveals that further improvement is required, new targets for genetic manipulation are identified followed by a new round of synthesis and analysis.
The Analysis-Genetic design-Synthesis cycle is repeated until the desired property is obtained. Metabolic engineering has been the subject of many reviews (Cameron and Tong, 1993; Nielsen, 2001; Stephanopoulos, 1999). In the following chapters I will focus on how this approach has been applied to generate xylose-utilising strains of S. cerevisiae. 
Pathways for Xylose Utilisation
Xylose utilisation is widespread among bacteria. The initial step in bacterial xylose utilisation is its isomerisation to xylulose by xylose isomerase (XI) (Hochster and Watson, 1954) (FIG. 1). Some species of yeast and filamentous fungi grow on xylose as the sole carbon source. Yeast and filamentous fungi first reduce xylose to xylitol using xylose reductase (XR) and thereafter xylitol is oxidised to xylulose by xylitol dehydrogenase (XDH) (Chiang and Knight, 1960) (FIG. 1).
S. cerevisiae was considered incapable of growth on xylose (Wang and Schneider, 1980) but grew on xylulose (Wang and Schneider, 1980) and produced ethanol from this substrate (Chiang, et al., 1981). It was later found that S. cerevisiae possesses enzymes with XR (Batt, et al., 1986; Kuhn, et al., 1995) and XDH (Batt, et al., 1986; Richard, et al., 1999) activities and consumed low amounts of xylose when co-metabolised with glucose, galactose or ribose (van Zyl, et al., 1989; van Zyl, et al., 1993). Xylulose utilisation suggests a link between xylulose and central metabolism. The S. cerevisiae gene XKS1 encodes the enzyme xylulokinase (XK) (Ho and Chang, 1989; Rodriguez-Pena, et al., 1998), which phosphorylates xylulose to xylulose 5-phosphate. In most organisms xylulose 5-phosphate is metabolised through the pentose phosphate pathway (PPP) (FIG. 2), but some bacteria, notably lactic acid bacteria, possess a phosphoketolase that cleaves xylulose 5-phosphate into glyceraldehyde 3-phosphate and acetyl phosphate. There have been reports on phosphoketolase activity in yeast (Whitworth and Ratledge, 1977; Evans and Ratledge, 1984; Ratledge and Holdsworth, 1985), but yeast probably also use PPP to metabolise xylulose 5-phosphate (Lighthelm, et al., 1988).
Although wild-type S. cerevisiae has enzymes that possess the activities of the Initial xylose-utilisation pathway, their activities are too low to allow xylose growth, and therefore S. cerevisiae has been transformed with heterologous genes encoding XI and XR/XDH. Xylose isomerases from several bacteria have been cloned and Introduced into S. cerevisiae (Ho, et al., 1983; Sarthy, et al., 1987; Amore, et al., 1989; Moes, et al., 1996), but only xyIA from the thermophilic bacterium Thermus thermophilus generated an active enzyme in S. cerevisiae (Walfridsson, et al., 1996). The transformant was able to consume three times more xylose than a control strain. XI from Thermus thermophilus has only trace activity at 30° C., while the highest enzymatic activity was obtained at 85° C.
XRs from different microorganisms have been characterised and they share a common feature in their preference for NADPH as a cofactor. The S. cerevisiae unspecific aldose reductase having XR activity (Kuhn, et al., 1995) and XR from Candida utilis (Bruinenberg, et al., 1983) exclusively use NADPH, while XR from P. stipitis (Verduyn, et al., 1985b; Rizzi, et al., 1988) and Candida tenius (Neuhauser, et al., 1997) Is also able to use NADH. The ratio of the specific activity of XR from P. stipitis using NADH and NADPH separately was approximately 0.65, regardless of the oxygen tension in the medium (Skoog and Hahn-Hägerdal, 1990). P. tannophilus produces two isoenzymes of XR, one of which can use both NADH and NADPH while the other is strictly NADPH dependent (Verduyn, et al., 1985a). Oxygenation-limitation favours the enzyme using both cofactors (VanCauwenberge, et al., 1989). Unlike XR, XDH from all microorganisms studied almost only uses NAD+ as cofactor (Bruinenberg, et al., 1983; Bruinenberg, et al., 1984; Richard, et al., 1999; Rizzi, et al., 1989b).
S. cerevisiae has been transformed with the P. stipitis genes XYL1 and XYL2 coding for XR and XDH, respectively (Kötter and Ciriacy, 1993; Tantirungkij, et al., 1993; Walfridsson, et al., 1995). The choice of P. stipitis as the donor organism was based on its capability to utilise NADH in the xylose reduction step. Ethanolic xylose fermentation with recombinant S. cerevisiae strains producing XR/XDH has resulted in low ethanol yield and considerable xylitol by-product formation. This has been ascribed to                (i) insufficient xylose transport,        (ii) unfavourable thermodynamics in the conversion of xylose to xylulose,        (iii) cofactor imbalance in the XR/XDH reactions and        (iv) an underdeveloped PPP.Xylose Transport        
S. cerevisiae does not have specific transporters for xylose, which is instead transported through facilitated diffusion by the hexose transporters. These have up to 100 times lower affinity for xylose than for glucose (Kotyk, 1967; Cirillo, 1968; Busturia and Lagunas, 1986; Kötter and Ciriacy, 1993), and therefore xylose is less efficiently transported into the cell when both glucose and xylose are present in the medium. In an anaerobic chemostat cultivation of the recombinant, xylose-utilising S. cerevisiae TMB 3001 using glucose and xylose in the feed, the specific uptake of xylose increased with lower dilution rate and higher xylose feed concentration. The residual glucose concentration decreased with lower dilution rate and a lower glucose concentration favoured xylose uptake. Xylose uptake was also less efficient than that of glucose during aerobic fermentation of S. cerevisiae TMB 3399 and 3400.
These results suggest that the low affinity for xylose could render the transport step significant control of the metabolic flux. However, one investigation demonstrated a 30 times higher transport capacity for xylose than the actual rate of xylose consumption (Kötter and Ciriacy, 1993), so the impact of xylose transport on the overall flux of xylose is still unclear.
Thermodynamics of Xylose Utilisation
The equilibrium constant for the reduction of xylose to xylitol has been estimated to be 0.575×103 at pH 7 (Rizzi, et al., 1988), and for the subsequent oxidation of xylitol to xylulose the equilibrium constant at pH 7 is 6.9×10−4 (Rizzi, et al., 1989a). Thus both reactions favour xylitol formation and the thermodynamics of the XDH reaction is unfavourable in the direction of ethanolic fermentation.
It is, however, inappropriate to consider a pathway thermodynamically unfeasible based on the presence of reactions with unfavourable equilibrium constants. According to the second law of thermodynamics, spontaneous processes occur in the direction that increases the overall disorder (or entropy) of the universe. A more convenient criterion for a thermodynamically feasible reaction is a negative Gibbs free energy (ΔG). Consider the single reaction:aA+bB→cC+dD or cC+dD−bB−aA=0
If we assume that biological systems are dilute solutions and therefore the fugacity and activity coefficients are equal to 1, Gibbs free energy for a chemical reaction, ΔG, is defined as:
      Δ    ⁢                  ⁢    G    =            Δ      ⁢                          ⁢              G        0              +          RT      ⁢                          ⁢              ln        ⁡                  (                                                                                          [                    C                    ]                                    c                                ⁡                                  [                  D                  ]                                            d                                                                                            [                    A                    ]                                    a                                ⁡                                  [                  B                  ]                                            b                                )                    
Hence ΔG is influenced by the standard Gibbs free energy (ΔG°) as well as the concentration of the metabolites involved in the reaction.
An algorithm has been developed to calculate the permitted metabolite concentration range where all reactions in a pathway have a negative AG and hence makes the pathway feasible (Mavrovouniotis, 1993). When this algorithm was applied to the reactions converting xylose into pyruvate using the pathway illustrated in FIG. 2 the reactions between dihydroxyacetone phosphate and 1,3-glyceraldehyde bisphosphate imposed the largest thermodynamic constraints. Since the same reactions were responsible for the largest thermodynamic constraints in glucose conversion to pyruvate (Mavrovouniotis, 1993) it was concluded that xylose conversion to pyruvate does not introduce new thermodynamic bottlenecks. The strongly favourable XR and XK reactions that take place before and after the XDH reaction compensated for the unfavourable thermodynamics of the XDH reaction.
Cofactor Imbalance
A cofactor imbalance arises from the fact that the XR reaction preferably consumes NADPH, while the XDH reaction exclusively produces NADH. When less NADH is consumed in the XR reaction, less NAD+ is available for the XDH reaction. Excess NADH generated in the XR/XDH reactions cannot be regenerated to NAD+ by the reaction catalysed by alcohol dehydrogenase (ADH), since this reaction oxidises the NADH formed in the glyceraldehyde 3-phosphate reaction (FIG. 2). If the amount of NAD+ is insufficient, xylitol accumulates and is excreted (Bruinenberg, et al., 1983).
XR and XDH have been subjected to protein engineering (Webb and Lee, 1992; Metzger and Hollenberg, 1995; Zhang and Lee, 1997; Kostrzynska, et al., 1998) to circumvent the cofactor imbalance. Site-directed mutagenesis was used in an attempt to alter the cofactor preference of XR from NADPH to NADH. The resulting enzyme lost 80-90% of its specific activity and the affinity for xylose decreased more than ten-fold (Kostrzynska, et al., 1998). The affinity for NADPH decreased, but remained constant for NADH. Attempts have also been made to alter the cofactor specificity of XDH towards NADP+ instead of NAD+. However, the affinity for NADP+ remained unchanged while the affinity for NAD+ decreased nine-fold and the specific activity of the mutated XDHs decreased to between 50 and 70% of that of the original enzyme (Metzger and Hollenberg, 1995).
The preferences for NADPH in the XR-catalysed reaction and NAD+ in the XDH-catalysed reaction, respectively, might have a thermodynamic origin. In cells of S. cerevisiae grown anaerobically on glucose, the ratio of NADPH to NADP+ is about 5, whereas the ratio of NADH to NAD+ is about 0.15 (Anderlund, et al., 1999). If these ratios are assumed to also be valid for xylose fermentation, using NADPH for xylose reduction and NAD+ for xylitol oxidation, the reactions catalysed by XR and XDH become thermodynamically feasible over a larger range of metabolite concentrations compared with the use of any other combination of cofactors in these reactions.
Metabolic flux analysis of S. cerevisiae TMB 3001 grown anaerobically in chemostat cultivation revealed a flexible utilisation of NADH and NADPH in the XR reaction. The flux of NADPH mediated xylose reduction decreased with increasing dilution rate, while the NADH mediated xylose reduction remained constant. The RNA and protein content of the biomass increased with the dilution rate, which leads to a higher NADPH-demand, and leaves less NADPH for the XR reaction. With increasing xylose concentration in the feed followed a higher flux of xylose reduction using NADH.
The relation in activities between XR, XDH and XK has been manipulated to decrease xylitol formation (Walfridsson, et al., 1997; Eliasson, et al., 2001). A kinetic model based on reported kinetic data for the three enzymes indicated an optimal XR:XDH:XK activity ratio of 1:10:4, which was also confirmed experimentally (Eliasson, et al., 2001). The model also showed that the NADH/NAD+ ratio strongly influenced the optimal ratio. On the other hand, the natural xylose-utilising yeast P. stipitis has higher XR activity than XDH activity under all levels of oxygenation and does not excrete xylitol even during anaerobiosis, (Lighthelm, et al., 1988). It has been demonstrated that P. stipitis strictly uses NADH for xylose reduction during anaerobiosis (Lighthelm, et al., 1988).
NADH is oxidised to NAD+ in the presence of an electron acceptor. When present, oxygen regenerates NAD+ in the electron transport chain. During oxygen-limited cultivation of S. cerevisiae TMB 3001, increased oxygenation led to an approximately constant yield of ethanol, while the yields of glycerol and xylitol decreased (Eliasson, 2001).
Addition of the external electron acceptors acetaldehyde (Ligthelm, et al., 1989) and acetoin (Bruinenberg, et al., 1983; Ligthelm, et al., 1989) to xylose fermentation by the naturally xylose-fermenting yeasts P. tannophilus and C. utilis regenerated NAD+ and prevented extracellular xylitol accumulation. Also in S. cerevisiae TMB 3001 anaerobic xylitol excretion decreased upon acetoin addition and, as with C. utilis, there was a marked increase in the acetate production (Bruinenberg, et al., 1983). Hence, the response of recombinant S. cerevisiae on acetoin addition was similar to that of natural xylose-utilising yeasts. Furfural, which is produced in the pre-treatment of lignocellulosic material (Larsson, et al., 1999) and has been shown to be an inhibitor of ethanolic fermentation by S. cerevisiae (Larsson, et al., 1999; Sanchez and Bautista, 1988; Taherzadeh, et al., 1999) also decreased xylitol formation.
MFA of acetoin addition to S. cerevisiae TMB 3001 cultivated anaerobically on xylose showed an increased flux to ethanol and that NADPH was produced in the conversion of acetaldehyde to acetate instead of in the oxidative PPP. As a consequence, more carbon is channelled through the lower part of glycolysis where ATP is produced. The ATP productivity increased from 0.9 mmol ATP (g biomass h)−1 to 1.8 mmol ATP (g biomass h)−1, but still no growth was detected. The interpretation of these results was that ATP is not limiting anaerobic growth on xylose of S. cerevisiae TMB 3001.
The Underdeveloped PPP
Recombinant S. cerevisiae, as opposed to the natural xylose-utilising P. stipitis, accumulates the intermediate sedoheptulose 7-phosphate when cultivated on xylose (Kötter and Ciriacy, 1993). It was suggested that S. cerevisiae has an underdeveloped PPP and especially insufficient transaldolase (TAL) activity. Overexpression of TAL gave better growth on xylose, but did not improve ethanol production (Walfridsson, et al., 1995). During anaerobic cultivation of S. cerevisiae TMB 3001 on a mixture of glucose and xylose, the flux between ribulose 5-phosphate and xylulose 5-phosphate was very low at all dilution rates and xylose concentrations. On glucose this reaction proceeds in the direction from ribulose 5-phosphate to xylulose 5-phosphate. Xylose utilisation without xylitol excretion requires this reaction to proceed in the opposite direction, from xylulose 5-phosphate to ribulose 5-phosphate. Overexpression of ribulose 5-phosphate-3-epimerase (RPE) in S. cerevisiae TMB 3001 did not increase the xylose utilisation (Johansson, 2001). Still, this enzyme deserves attention. The S. cerevisiae RPE has a Km value of 1.5 (Bär, et al., 1996)-2.4 (Kiely, et al., 1973) mM for ribulose 5-phosphate, but there are no reports on its Km value for xylulose 5-phosphate. If RPE in S. cerevisiae has a significantly lower affinity (=higher Km) for xylulose 5-phosphate than for ribulose 5-phosphate, this would mean a lower specific activity to convert xylulose 5-phosphate to ribulose 5-phosphate and thus decreased xylose utilisation.
Natural xylose utilising organisms might very well have RPEs with higher affinities for xylulose 5-phosphate than the RPE of S. cerevisiae. 